Thymidylate synthase (TS) catalyzes the conversion of dUMP and 5, 10- methylenetetrahydrofolate (CH2/H/2 folate) to dTMP and 7,8-dihydrofolate. It is essential for de novo synthesis of dTMP and consequently is required for DNA synthesis. Continued in-depth studies of this enzyme are important (i) for increasing the knowledge base of TS, an important chemotherapeutic target, especially in cancer chemotherapy, (ii) as a paradigm for related enzymes, (iii) as a model for enzyme structure/function studies, and (iv) as a platform for technology development. In the present proposal we plan: (i) studies on the mechanism and inhibition of TS, (ii) site directed mutagenesis studies to understand functions of important amino acid residues, (iii) combinatorial mutagenesis of the dimer interface, and iv) attempts to evolve the enzyme into a more effect catalyst by gene-shuffling, or molecular evolution. Several inhibitor studies will be performed, including a search for peptides and small molecules that may inhibit subunit association. Mutagenesis will be accomplished primarily by cassette multiple mutagenesis of a synthetic L. casei Ts gene; in addition, experiments in combinatorial mutagenesis and gene shuffling are proposed. Mutant enzymes will be purified and subjected to detailed characterization in order to determine the effects of amino acid replacement at key positions. We will carry out X-ray crystallographic analysis of interesting mutant enzymes, and enzyme-inhibitor complexes in collaboration with Dr. Robert Stroud, UCSF. The search for novel small molecule inhibitors will be carried out using the program DOCK in collaboration with Dr. Irwin Kunts, UCSF. Some mechanistic question will be approached using NMR spectroscopy in collaboration with Dr. Thomas James, UCSF.